Nature Methods 3, 281 - 286 (2006)
Published online: 22 March 2006; | doi:10.1038/nmeth866
Genetically encoded fluorescent indicator for intracellular hydrogen peroxideVsevolod V Belousov1, Arkady F Fradkov1, Konstantin A Lukyanov1, Dmitry B Staroverov1, 2, Konstantin S Shakhbazov3, Alexey V Terskikh3, 4
& Sergey Lukyanov11
Laboratory of Genes for Regeneration, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street 16/10, Moscow 117997, Russia. 2
Evrogen, JSC, Miklukho-Maklaya Street 16/10, Moscow 117997, Russia. 3
School of Life Science, Swiss Federal Institute of Technology, Lausanne (EPFL), CH-1015 Lausanne, Switzerland. 4
The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, California 92037, USA.
Correspondence should be addressed to Sergey Lukyanov luk@ibch.ru We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H2O2) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H2O2-sensing protein, OxyR. Using HyPer we monitored H2O2 production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H2O2 occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (  ) and a change in cell shape. We also observed local bursts in mitochondrial H2O2 production during oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H2O2 increase upon physiological stimulation. Using HyPer we detected temporal increase in H2O2 in the cytoplasm of PC-12 cells stimulated with nerve growth factor.
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